Protein and Peptide Mass Spectrometry in Drug Discovery (Gross/Protein Mass Spec Drug Discovery) || Ion Activation and Mass Analysis in Protein Mass Spectrometry

CHAPTER 2

Ion Activation and Mass Analysisin Protein Mass Spectrometry

CHENG LIN and PETER O’CONNOR

In this chapter we consider the various methods of activation that can be used to

fragment peptide and protein ions and thereby be used to determine their amino-acid

sequences. After a brief introduction to the terms important in mass spectrometry

(MS) analysis, we describe the methods used to activate peptide and protein ions for

sequencing by MS. This section is followed by a discussion of mass analysis,

particularly as it applies to the MS/MS experiment.

2.1 INTRODUCTION

A mass analyzer is the heart of a mass spectrometer, where ions are separated

according to their mass-to-charge ratios (m/z). Althoughm/z has a dimension of mass

over charge, it is often expressed as a dimensionless number in the MS literature,

where the mass is measured in the unified atomic mass unit, u, or dalton (Da), 1

u� 1.66� 10�27 kg, and the charge is measured as number of elementary charges, e,

1 e� 1.602� 10�19 coulombs. The performance of a mass analyzer is characterized

by a number of parameters, including its mass accuracy, mass resolving power, mass

range, scan speed, and tandem MS analysis capability.

2.1.1 Mass Accuracy

Mass accuracy describes the ability of the mass analyzer to measure the correct mass

(m/z) of an ion, and precision is a measure of the ability to reproduce the mass

measurement. Mass accuracy may be expressed as an absolute number, typically in

mDa’s, representing the difference between the theoretical and the measured masses.

Protein and Peptide Mass Spectrometry in Drug Discovery, Edited by Michael L. Gross, Guodong Chen,and Birendra N. Pramanik.� 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

43

It is also frequently given as the relative ratio of this mass difference to the theoretical

mass value, in parts per million (ppm). Mass accuracy is closely related to the mass

resolving power of themass analyzer. For example, a low resolving power instrument,

such as a quadrupole ion trap (QIT) mass spectrometer, can only provide a typical

mass accuracy of approximately 100 ppm, whereas a high-end Fourier-transform ion

cyclotron resonance (FTICR) mass spectrometer can routinely achieve a mass

accuracy in the sub-ppm range. Other factors that affect the mass accuracy include

the stability of the instrument, mass calibration, and the peak centroid determination.

2.1.2 Mass Resolving Power

Mass resolving power is the ability of a mass analyzer to separate ions with closely

spaced m/z values. For an isolated peak the mass resolving power (RP) can be

calculated using the formula

RP ¼ m=z

Dðm=zÞFWHM

; ð2:1Þ

where D(m/z)FWHM is the full width of the peak at its half maximum, but it can also be

substituted by the peak width at other fractions of the peak maximum.Mass resolving

power may also be calculated using adjacent overlapping peaks. In this definition the

D(m/z)FWHM of equation 2.1 is replaced by Dm, or the mass resolution, which is the

smallest mass difference between two equal magnitude peaks so that the valley

between them is a specific fraction of the peak height. For Gaussian shaped peaks, a

50% valley exists when Dm is approximately 141% of the D(m/z)FWHM value. An

immediate consequence of poor mass resolving power is the inability to determine

the peak position accurately in the presence of nearby peaks. Figure 2.1 illustrates the

effect of mass resolving power on the obtainable mass accuracy. For two Gaussian

shaped peaks (Figure 2.1) of equal height for ions of m/z 1000 and 1001, a mass

resolving power of 1000 results in close to a 100-mDa difference between the

observed and actual peak positions (Figure 2.1B), whereas a slight increase of RP

to 1410 improves the mass accuracy to around 4mDa (Figure 2.1A). This overlapping

problem is more severe when the nearby peak is of a higher intensity than the peak of

interest. When the “interfering” peak atm/z 1000 is five times as intense as the one of

interest atm/z 1001 and the RP is still 1410, the peak position of the latter is shifted by

26mDa (Figure 2.1C); when that ratio increases to 10, the valley disappears, and the

m/z 1001 peak cannot be identified in the spectrum (Figure 2.1D).

2.1.3 Mass Range

The mass range of a mass analyzer is the range of m/z values an ion can have to be

detected. Quadrupole mass analyzers, magnetic sectors, and quadrupole ion traps can

typically scan up to around m/z 4000, whereas FTICR mass analyzers can easily

detect ions of m/z value over 10,000. A linear time-of-flight (TOF) analyzer has no

upper mass limit in principle, but the practical upper mass limit of a reflectron TOF

instrument is approximately 10,000; the limit is due to the tendency of large

44 ION ACTIVATION AND MASS ANALYSIS IN PROTEIN MASS SPECTROMETRY

biomolecules to undergo postsource decay (PSD) and not be detectable in a reflectron-

based instrument because the precursor ions are lost to fragmentation, and the product

ions are not refocused. Low mass limits also exist for some mass analyzers. For

example, in both FTICR and Orbitrap instruments, the lowestm/z value detectable is

limited by the sampling frequency applied, as determined by the Nyquist theorem,

with typical values of 50 to 200Da when the B field is large (e.g.,44.8 T). The mass

range of a mass spectrometer may be further reduced by factors not determined by the

mass analyzer. For example, multipole ion guides commonly used for ion transfer

have their own mass cutoffs.

2.1.4 Scan Speed

Scan speed describes how fast a mass analyzer can acquire mass spectra. For

scanning mass analyzers such as quadrupoles, QIT’s, and sector instruments, this is

literally the speed at which a mass analyzer scans through a certain m/z range. For

other types of instruments (e.g., FTICR and orbitrap mass analyzers), ions of allm/z

values are detected simultaneously, in which case the scan speed may be defined as

the rate at which each individual mass spectrum is acquired. ATOF analyzer is the

fastest analyzer, capable of acquiring thousands of spectra in one second. An FTICR

mass spectrometer, on the other hand, is often operating at a much slower rate,

taking a second or longer to acquire one high-resolving-power mass spectrum. Scan

FIGURE 2.1 The impact of mass resolving power (R.P.) on the mass accuracy for two peaks

separated by 1Da. The arrow indicates the measured peak position of the m/z 1001 ion at the

presence of another ion atm/z 1000. (A) R.P.¼ 1410, equal peak heights; (B) R.P.¼ 1000, equal

peak heights; (C) R.P.¼ 1410, the peak height ratio is 5:1; (D) R.P.¼ 1410, the peak height ratio

is 10:1.

INTRODUCTION 45

speed is particularly important when the mass analysis is performed in conjunction

with online separation techniques such as high-performance liquid chromatography

(HPLC) or ionmobility, where analytes of interest are eluting only for a short period

of time. For a given mass analyzer, there is often a trade-off between scan speed and

mass resolving power, and accuracy.

2.1.5 Tandem MS Analysis

Tandem MS analysis refers to the process where a selected ion of interest (called the

precursor ion) is isolated and dissociated to generate fragment ions whosem/z values

are then measured. The masses of the fragment ions can be used to elucidate the

structure of the precursor ion or, as is relevant to this book, sequence peptides and

proteins. TandemMSexperimentsmay be performed tandem in space, which requires

the use of two separate, physically distinct mass analyzers, such as those done in a

triple quadrupole instrument or in a TOF-TOF mass spectrometer. It may also be

performed tandem in time, in which case isolation of the precursor ion and mass

analysis of the fragment ions are achieved using the same mass analyzer, but the

events of isolation, activation, and analysis are separated in time. This is usually done

in trap instruments, such as a QIT or an FTICR mass spectrometer.

Tandem MS analysis may be performed once (MS/MS), or multiple times

consecutively, with each of theMS/MS experiments done on a fragment ion generated

in the previous MS/MS step (this is known as theMSn experiment). MSn experiments

produce feature rich fingerprints of the precursor ion by providing detailed structural

information on each of the isolated fragment ions from the product-ion spectrum

acquired in an MSn�1 experiment, making them a valuable tool in metabolite

identification in drug discovery. In addition, when a product-ion spectrum is domi-

nated by just a few fragments resulting from facile cleavages, MS3 experiments are

often needed to generate more complete structural information of the precursor ion.

MSn experiments are also used to characterize carbohydrate structural isomers, based

on sequential losses of different derivatized monosaccharide units that carry frag-

mentation “scars” throughout the MSn tree [1]. MSn (n4 2) experiments can best be

performed in trapping instruments.

In tandemMS experiments, it is usually necessary to activate the precursor ion first

to induce fragmentation. Ion activation can be achieved in many ways: via collisions

with gases or surfaces, absorption of IR or UV photons, or activation by ion–electron

interactions [2].

2.2 ION ACTIVATION AND TANDEM MS ANALYSIS

2.2.1 Introduction: Fragmentation in Protein MS

Before we get into the details of various ion activation methods, it is helpful to look at

what fragment ions may be produced in tandem MS experiments of peptide ions, and

how this information can be used for their structural characterization. Throughout the


discussions in this section, the term peptide will be used in place of peptide/protein, as

the two share similar fragmentation behavior. Figure 2.2 illustrates the common

types of fragment ions observed in the tandemmass spectrum of a tetra-peptide ion [3].

These can be classified into twobroad categories: the backbone fragment ions (a-, b-, c-,

and x-, y-, z-ions),which result from the cleavage of a backbone bond, shownon the top

panel; and the secondary side-chain fragment ions, or satellite ions (d-, v-, andw-ions),

which are often generated by radical-driven, charge-induced, or even charge-

remote fragmentations of the backbone fragment ions, shown on the bottom panel

of the scheme.

Backbone fragment ions are also referred to as sequence ions because they are

useful in peptide sequencing. Gas-phase peptide sequencing is based on the knowl-

edge that adjacent backbone fragment ions of the same series are spaced by themasses

of the amino-acid residues. For example, themass difference between the bn and bnþ 1

ion is the mass of the nth amino-acid residue, and that mass can be used to deduce the

identity of the amino acid, with the exception of isomeric amino acids. Secondary

side-chain fragment ions, on the other hand, contain important information on the

identity of the side chain; these fragments are particularly useful for differentiation of

isomeric amino-acid residues, (e.g., leucine and isoleucine). Other types of ions may

also be produced in tandem MS experiments, including the immonium ions, internal

fragment ions, and ions resulting from side-chain or small-molecule losses. Although

these ions are not always structurally informative, and their presence can make the

interpretation of the mass spectra more difficult, they can be useful (e.g., immonium

ion formation and side-chain losses from the molecular ion are often used to identify

the existence of certain amino-acid residues in the peptide).

Finally, because proteins undergo extensive post-translational modifications

(PTMs), protein characterization should include the identification and location of

PTMs in addition to the sequence determination. A PTM can be identified by the

observation of the characteristic mass shift in molecular ions; however, PTM site

FIGURE 2.2 (Top panel) Nomenclature for sequence ions in peptide tandem mass spectra,

as first proposed by Roepstorff and Fohlman (Roepstorff P., Fohlman J., Biomedical Mass

Spectrometry, 1984, 11, 601); (bottom panel ) structure of satellite ions.

ION ACTIVATION AND TANDEM MS ANALYSIS 47

location usually requires tandem MS experiments. Thus it is advantageous to retain

PTMs during ion activation and backbone bond breakage so that the PTMmass “tags”

the fragment ions to which it is attached, allowing successive localization in further

stages of MSn.

2.2.2 Collisional Activation Methods

Collisionally activated dissociation (CAD), or collision-induced dissociation (CID),

is by far the most commonly applied ion activation method in tandem MS analysis.

In a CAD experiment the precursor ion is allowed to collide with neutral gas

molecules, resulting in energy transfer and ultimately internal excitation of the

precursor ion [4–6]. Collisional activation can be achieved with a single high-energy

(typically41000 eV) collision, or with many low-energy (51 to 100 eV) collisions.

Low-energy CAD is usually implemented in trapping instruments; examples are

linear trapping quadrupoles (Q-CAD), quadrupole ion traps, and FTICR mass

spectrometers (as in sustained off-resonance irradiation, or SORI-CAD) [7–10].

Although the peptide ion has been accelerated to a kinetic energy upward to 100 eV in

the laboratory frame, it is the collisional energy in the center-of-mass frame (ECOM)

that determines the maximum amount of energy that can be transferred to excite an

ion’s internal ro-vibrational modes. Because commonly used collision partners (e.g.,

He, N2, or Ar) are much lighter than a typical peptide ion, ECOM is often orders of

magnitude smaller, as calculated by equation 2.2, wherem is the mass of the collision

gas, M is the mass of the ion to be activated, and ELab is the laboratory energy:

ECOM ¼ m

mþMELab: ð2:2Þ

Thus, a heavier collision gas such asAr orN2 is frequently used in low energyCAD,

as these gases allow a more efficient transfer of energy than does the lighter helium.

Low-energy CAD is generally considered an “ergodic” or “slow-heating”

fragmentation method, where the term “slow” is used relative to the rate of intra-

molecular vibrational energy redistribution (IVR). In low-energy CAD experiments

ion activation is achieved via multiple collisions, each depositing a small amount of

energy into the precursor ion. Because the bond dissociation is preceded by the energy

randomization, fragmentation rarely occurs at a site where the energy was first

deposited in the collision. Instead, when the overall energy of the ion is raised above a

certain dissociation threshold (activation barrier), fragmentationmay occur, typically

resulting in the rupture of theweakest bondwithin themolecule. For peptide ions, this

is usually the amide bond, leading to the formation of b- and y-ions.

Given that direct amide bond cleavage requires the precursor ion to be excited

to a substantially higher level than typically achievable in a low-energy CAD

experiment, a “mobile proton” model can explain the b/y fragmentation pathway

(Scheme 2.1) [11,12]. In essence, the fragmentation is initiated by the attachment or

movement of a mobile proton to the oxygen or the nitrogen of the amide bond to be

cleaved (the scheme shows the proton attachment to the amide nitrogen); the proton

attachment not only weakens the amide bond but also increases the electrophilicity of


the adjacent carbonyl, making it more susceptible to nucleophilic attack by either the

amide oxygen (the oxazolone pathway, Scheme 2.1A) or the nitrogen (the diketopi-

perazine pathway, Scheme 2.1B) of its N-terminal neighbor. Thus peptide fragmenta-

tion proceeds via a low-energy rearrangement reaction, initiated by proton transfer,

rather than direct accumulation of vibrational energy at an amide bond to induce its

rupture. Because an arginine residue had the tendency to sequester a proton (it has high

proton affinity), peptide ions with the number of arginine residues equal to or higher

than the number of charges require higher collision energy to fragment. Although, in

general, CAD cleaves the amide bonds relatively indiscriminately, selective cleavages

are observed near particular amino-acid residues. For example, cleavageN-terminal to

the proline residue is often enhanced, whereas cleavage C-terminal to the proline

residue is suppressed. This “proline effect” may be due to its relatively high gas-phase

proton affinity, which facilitates its amide nitrogen protonation, and to the hindered

formation of an oxazolone b-ion containing the proline residue as itsC-terminus owing

to a strained bicyclic structure. Enhanced cleavage is also observed C-terminal to a

protonated histidine residue, likely owing to its ability to transfer a proton to the

backbone, allowing the side-chain nitrogen to attack the carbonyl and to form a

resonance-stabilized cyclic b-ion. Finally, when no mobile proton is available,

preferential cleavage at acidic residues occurs, with the acidic H of the aspartic or

glutamic acid side chain serving to initiate cleavages at its C-terminal side.

High-energy CAD experiments are usually performed in magnetic sector [13,14]

or TOF-TOF mass spectrometers [15] (beam instruments) where ions can be easily

accelerated to have lab-frame translational energy of several thousand eV.Helium gas

is the preferred collision partner in the high-energy CAD experiment, as it minimizes

the scattering losses of both the precursor and the product ions; scattering is more

severe in beam instruments because higher collision energy is employed, and focusing

SCHEME 2.1 Mobile proton models for b/y cleavages. (A) the oxazolone pathway; (B) the

diketopiperazine pathway.


methods are sparse. These focusing methods are available when collisions are carried

out in a multiple ion guide/trap. In high-energy CAD spectra, in addition to the b- and

y-type ions and/or small-molecule losses, abundant immonium ions, internal ions

and secondary fragments such as d- and w-type ions are also readily produced, the

latter of which provide useful information for side-chain differentiations [13,16].

Despite its wide implementation, CAD also has several drawbacks. One of its

major limitations is its poor applicability in PTM analysis. Many PTMs are more

labile than the backbone amide bond and are the first to fall off (whether via direct

scission or a rearrangement is not clear) when the ions are collisionally activated; this

makes PTM location a challenging task. In addition, when a labile group is present in a

peptide or protein, the CAD spectrum is often dominated by a fragment ion produced

by loss of the PTM. This loss preempts peptide bond cleavages, causing fewer

backbone fragments to form. Likewise, when a particularly labile dissociation

channel exists, such as the b/y cleavage at theAsp-Pro sequence or loss of a phosphate,

other fragmentation channels may also be suppressed, resulting in poor sequence

coverage. Furthermore, sequence scrambling can occur in CAD experiments, in

which an oxazolone b-ion can cyclize and reopen at different position; such processes

give a product that, upon further activation, can produce misleading sequence

ions [17,18]. The use of a collisional gas in CAD can compromise the high vacuum

of the spectrometer, and the gas may need to be pumped away before mass analysis,

particularly in an FTICR instrument. The pump-down time results in longer spectral

acquisition time and reduced throughputs. AdditionallyCADhas limits in quadrupole

ion traps and linear ion traps because the resonant excitation raises the low-mass

cutoff of the instrument (discussed below in Section 2.3).

Alternatively, collisional activation can be achieved by ion/surface collisions

without the use of collision gases, as implemented in surface-induced dissociation

(SID) [19,20]. In general, SID produces product-ion spectra that are similar to those

generated by CAD. Higher ratios of a- to b-ions and enhanced immonium-ion

formation, however, also occur, and this is indicative of increased access to higher

energy and secondary fragmentation channels. Unlike low-energy CAD, ion activa-

tion in SID is achieved in a single collision, rather than being slowly heated via

multiple collisions until the dissociation threshold is reached. The effective neutral

partner mass of the surface in SID (m in equation 2.2) is also much higher than that of

collisional gases, leading to a higher center-of-mass collision energy available for ion

excitation. The efficiency of translational-to-internal energy conversion for SID

depends on the kinetic energy, the size of the precursor ions, and the nature of the

surface. The most commonly used surface is metallic with a nonconducting fluori-

nated, self-assembled monolayer (SAM) to minimize ion neutralization. SID can be

implemented in a variety of mass spectrometers, including the tandem quadrupole,

TOF-reflectron, Q-TOF, and FTICR instruments [21–23].

2.2.3 Photodissociation

Peptide ions may be optically excited as well. Photodissociation (PD) is best applied

to trapped ions to allow for sufficient ion/photon interaction time, hence indicating as


instruments linear ion traps [24] or FTICR mass spectrometers [25], although

photodissociation in TOF/TOF instruments [26] can also be useful. Photodissociation

offers several advantages over CAD, including ease of implementation, better control

of energy inputs, and selectivity based on the absorption spectra of precursor ions. For

photodissociation in an FTICR instrument, the gas-free operation can dramatically

reduce the MS/MS spectral acquisition timewithout deteriorating the vacuum, which

is needed for high-resolving-power mass analysis. Compared to SORI-CAD experi-

ments, which excite ions over a narrow m/z window over time, all precursor ions of

interest and in different charge states are dissociated simultaneously in a single

photodissociation event. For photodissociation in a linear or quadrupole ion trap, there

is no low-mass cutoff because no translational excitation of the precursor ions is

involved, and removing the need for translational activation also minimizes scatter-

ing. Finally, because all product ions are produced either on axis (as in linear ion traps)

or in the center of the trap (as in FTICRs), MSn experiments can be easily performed.

Infrared Multiphoton Dissociation The photons may originate from back-

ground blackbody irradiation, as in the blackbody infrared radiative dissociation

(BIRD) experiment [27], or from a laser, with wavelengths ranging from the mid-

infrared (IR) region to the vacuum ultraviolet (VUV) end of the spectrum. The most

commonly used IR laser is the continuous wave (cw) CO2 laser operating at 10.6 mm,

which vibrationally excites peptide ions, for example. Given that a 10.6-mm photon

has an energy of around 0.117 eV, or 11.3 kJ/mol, whereas a typical chemical bond has

a bond dissociation energy (BDE) of around 400 kJ/mol, absorption of hundreds or

even thousands of IR photons is necessary before fragmentation occurs. Thus, as for

low-energyCAD, infraredmultiphoton dissociation (IRMPD) also “heats” slowly the

ionswith IVRpreceding bond dissociation.As a result IRMPDof peptide ions yields a

fragment pattern similar to that of CAD, with the exception that, because fragment

ions from IRMPD can continue to absorb photons and further fragment, secondary

fragmentation is enhanced, for good or bad.

The IRMPD efficiency can be improved by covalently attaching to the peptides an

IR-chromophore, such as a phosphonite or sulfonate group. N-terminal sulfonation

affects the CAD and IRMPD spectra of the peptide YGGFLR; the spectrum was

acquired in a linear ion trap (Figure 2.3) [28]. The sulfonate group increases the

photoabsorptivity of the peptide at 10.6mm and leads to extensive fragmentation at a

shorter irradiation time. Furthermore the negative charge of the sulfonate neutralizes

the N-terminal fragment charge, greatly simplifying the product-ion mass spectrum.

Compared to the complex CAD spectrum showing both N- and C-terminal and other

fragment ions, the IRMPD spectrum of the modified peptide is dominated by a y-ion

series that makes de novo sequencing amuch easier task. Finally, the low-mass cutoff

in the CAD spectrum prevents observation of the y1-ion, whereas a complete series of

y-ions are formed upon IRMPD.

Ultraviolet Photodissociation UV lasers can also be used to fragment peptide

ions in a variety of mass spectrometers, including the linear ion trap, tandem TOF,

tandem sector, and FTICR instruments [26,29–32]. Unlike IR lasers, a UV laser


excites the peptide ions electronically. Electronic excitations are “vertical” excita-

tions that occur on a femtosecond time scale, shorter than a vibrational period.

Moreover, a UV photon contains much higher energy than an IR photon (e.g., a

193 nm photon has an energy of�620 kJ/mol, enough to break most covalent bonds).

Therefore, it is possible for dissociation induced by a single UV photon absorption to

occur rapidly prior to the IVR. As such, UV photodissociation may produce spectra

that are dramatically different from and complementary to either low-energy CAD or

IRMPD spectra. Fast absorption of high energy also permits the UVPD method to be

coupled to beam-type instruments.

CommonUV lasers employed to fragment peptide ions include excimer lasers and

various harmonics of the Nd:YAG laser. The 266 nm light (the third harmonic of the

Nd:YAG laser output, or Y4) is absorbed strongly by the side chains of tryptophan,

tyrosine, and phenylalanine and fragments peptide ions containing these chromo-

phores [32]. A near-UV chromophore may also be covalently or noncovalently

attached to the peptide ions, which enables photodissociation at longer wavelengths,

such as the 355-nm light from the Nd:YAG laser (the second harmonic, Y3) [33]. In

either case, as the absorption occurs locally at specific chromophores, the energymust

be redistributed before extensive backbone fragmentations may occur, which results

in a general fragmentation pattern similar to that observed in CAD, with enhanced

fragmentation near the chromophores. Unusual fragmentations generating radical

FIGURE 2.3 ESI-MS/MS mass spectra of YGGFLR. (A) CAD of unmodified peptide;

(B) CAD of N-terminal sulfonated peptide; (C) IRMPD of N-terminal sulfonated peptide.

Magnification scales apply to all spectra along themass range indicated.Adaptedwithpermission

from American Chemical Society (Wilson J. J., Brodbelt J. S., Anal. Chem. 2006, 78, 6855).


products sometimes occur, and are attributed to direct fragmentation near the

chromophores.

A second UVPD approach is to choose a wavelength absorbed by a universal

chromophore such as the backbone amide bond. The amide bond has several UV

absorption bands centered at near 190 nm and near 160 nm, both of which are readily

accessible with excimer lasers (ArF: 193 nm, F2: 157 nm) [34]. Of the two wave-

lengths, the 193 nm is more convenient, primarily because it can travel through air

with only small absorption [35]. Light of 157 nm must be transmitted either through

vacuum or an inert-gas-protected environment because it is strongly absorbed by

oxygen in the air. Figure 2.4 shows a typical UVPD spectrum of a tryptic peptide

containing arginine at itsC-terminus; the spectrum can be acquired by using either the

193- or the 157-nm light in a tandemTOF instrument [34].Unlike IRMPDorUVPDat

longer wavelengths, UVPD of peptide ions at these two wavelengths leads to

extensive a/x cleavages and other secondary fragmentation. UV absorption at these

FIGURE 2.4 Tandem-TOF photodissociation of Glu-Fibrinopeptide B (EGVNDNEEGFF-

SAR)using (A) 193 and (B) 157 nm light. Reproducedwith permission fromElsevier (Thompson

M. S., Cui W., Reilly J. P., J. Am. Soc. Mass Spectrom. 2007, 18, 1439).


wavelengths apparently results in the homolytic cleavage of the Ca�C(¼O) bond,

producing two radical species a þ 1 and x þ 1, both of which may either lose a

hydrogen to form the a- or x-ions, depending on the location of the charge carrier(s), or

undergo secondary radical-induced rearrangements to form d-, w-, and v-type ions.

Some b- and y-ions are also seen in the UVPD spectra, particularly when the arginine

residue is replaced by a lysine. This may be due to the higher mobility of a proton

associated with the charged lysine residue, and that mobile proton facilitates the low-

energy fragmentation processes. Finally, because UVPD may occur prior to energy

randomization, a distinct advantage of UVPD over CAD and IRMPD is its ability to

retain labile PTMs, such as phosphorylations and glycosylations, while backbone

bonds break. Although this possibly non-ergodic or nonstatistical behavior may

pertain to many electron-induced dissociation methods discussed in the next section,

UVPD offers a unique benefit in that it is applicable to singly charged ions generated

by MALDI, as it does not involve charge reduction.

Femtosecond Laser-Induced Dissociation Very recently a new tandem MS

technique termed the femtosecond laser-induced ionization/dissociation, or fs-LID,

was developed in which an ultrafast femtosecond laser with high peak power

(41013W/cm2) and high repetition rate (kHz) fragments peptide ions in an ion trap.

Although the fs laser is often a Ti:Al2O3 laser operated in the near IR region

(�800 nm), the fs-LID spectra are very different from typical IRMPD spectra, as

exemplified in Figure 2.5 [36]. In addition to the b- and y-ions associated with ergodic

fragmentation pathways, abundant a-, c-, x-, and z-ions are produced along with

secondary fragment ions. Phosphate groups are retained in many fragment ions

produced by peptide-bond cleavages, and these ion series can be used to locate the

FIGURE 2.5 fs-LID MS/MS (200 msec irradiation) of the [M þ H]þ precursor ion of

GAILpTGAILK. Adapted with permission from American Chemical Society (Kalcic C. L.,

et al., J. Am. Chem. Soc. 2009, 131, 940).


phosphorylation site [37]. The mechanism of fs-LID involves tunneling ionization of

the precursor ion in the presence of the strong electromagnetic field produced by the

high-power femtosecond irradiation, which generates a radical species that may

undergo fragmentation induced by vibrational or electronic excitation. This hypoth-

esis is supported by the presence of a doubly charged radical species seen in the fs-LID

spectrum of a singly charged precursor ion. Like the shorter wavelength UVPD, fs-

LID also produces more nonstatistical fragmentations and is amenable to singly

charged precursor ions. This approach complements conventional CAD, IRMPD, and

electron-induced dissociation methods (next section).

2.2.4 Electron-Induced Dissociation

A third method to activate peptide ions is through ion–electron or ion–ion interac-

tions. Electron-induced dissociation has been around for many decades, but it was not

until the late 1990s with the implementation of electron-capture dissociation (ECD)

that it found broad applications in the structural analysis of biomolecules [38–41].

The first ECD spectrum was actually acquired during a UVPD study in an FTICR

instrument, where a misaligned 193-nm laser beam hit the ICR trap surface,

generating photoelectrons that induced ECDof peptide ions. Since then, conventional

electron sources (e.g., a directly heated filament, an indirectly heated dispenser

cathode, or a cold field emitting device) are being used instead of the laser.

ECD spectra of multiply charged protein ions are usually dominated by the c- and

z-ion series resulting from the N–Ca bond cleavage [38]. Preferential cleavage of

disulfide bonds in ECD also occurs [42]. An important characteristic of ECD is its

ability to generate extensive backbone cleavages while leaving the more labile PTMs

and even noncovalent interactions intact [39–41,43–45]. The cause of this putative

nonstatistical behavior of ECD is the center of an ongoing debate on the primary ECD

mechanism. Some propose that ECD is a non-ergodic process initiated by the electron

capture at a charge site, followed by hydrogen transfer to the backbone carbonyl

inducing N�Ca bond cleavages. Others argue against the non-ergodic premise and

propose that the electron capture first occurs at the backbone carbonyl, generating an

anion-radical super base stabilized by a remote charge; the newly formed species then

undergoes facile N�Ca bond cleavage prior to proton transfer, leading to the

formation of c- and z-ions. A general mechanism (Scheme 2.2) proposes that the

electron capture puts the peptide ion in a Rydberg state (with �4–6 eV of excess

energy owing to recombination of opposite charges) that may sample a number of

electronic states of the charge-reduced ion as it “rattles” down the energy ladder. Both

mechanisms could be at work, depending on the electronic state of the peptide ions

from which the dissociation occurs [46].

ECD is highly complementary to the conventional CAD method, as illustrated by

Figure 2.6, which is a heat map showing the frequency of fragmentation occurrence as

a function of the neighboring amino acid residues [47]. One feature that stands out is

that whereas cleavageN-terminal to proline is enhanced in CAD, it is rarely observed

in ECD, because the N�Ca bond cleavage at the proline site still leaves the N- and

C-terminal “fragments” connected by a covalent bond owing to proline’s ring


structure. Figure 2.6 suggests that it is often advantageous to perform ECD and CAD

as a duet to take advantage of the complementary nature of the two methods.

Moreover, obtaining both the ECD and CAD spectra of the same sample increases

the confidence of peak assignment by utilizing the “golden pair,”† and consequently

lead to more reliable protein identifications in both database searching method and

de novo sequencing [48].

It is worth noting that N�Ca bond cleavage may also occur on the N-terminal side

of the affected carbonyl. Although this is generally disfavored because the N-radical

that is formed is unstable, it may be important when unusual amino-acid residues are

involved. For instance, Ca-Cb cleavage at the isoaspartic acid residue site will lead to

the formation of c þ 57 and z� 57 diagnostic ions for the differentiation of aspartic

and isoaspartic acid residues; this differentiation is an important goal in protein

deamidation studies [49,50].

SCHEME2.2 Generalmechanism for ECD in ground and excited electronic states of peptide

ions. Reproduced with permission from Elsevier (Syrstad E. A., Turecek F., J. Am. Soc. Mass

Spectrom. 2005, 16, 208).

† The “golden pair” refers to a b- (or y-) ion from the CAD spectrum that is also observed with a correlated

c- (or z-) ion from the ECD spectrum. These pairs allow determination of the directionality of the cleavage,

thus inherently labeling the N-terminal and/or C-terminal fragment ion series.


In ECD, the initial N�Ca bond cleavage produces an even-electron c ion and an

odd electron z. ion. The radical on the alpha carbon of the z. ion may propagate via

radical-driven rearrangements, including hydrogen transfer between the c and

z species before they separate to produce a radical c. ion and an even-electron

z ion [51–53]. Hydrogen abstraction within the z. fragment can lead to the formation

of secondary w ions, internal fragments, or z-ions with additional partial or complete

side-chain losses [54–56]. This free-radical cascade may explain the formation of

backbone fragments in cyclic-peptide ECD, where the capture of a single electron

results in multiple backbone cleavages [57].

The ECD efficiency and sequence coverage can often be improved by vibrational

excitation of the precursor ions, particularly for larger proteins or peptides with

extensive noncovalent interactions [58] such as those between a phosphate group of a

phosphopeptide and other positive charges [59]. The vibrational excitation breaks

noncovalent bonds that hold the nascently formed c and z fragments together and

facilitates formation of individual fragment ions. Furthermore there may be an

increase in the conformational heterogeneity of the precursor ion, leading to a more

extensive fragmentation pattern or increased secondary fragmentation. Ion activation

can be achieved via collisions with gases as done in plasma ECD [60] via IR

irradiation, as implemented in the activated ion or AI-ECD [58], or simply by

increasing the electron energy as employed in hot ECD [61,62].

The success of ECD has led to the implementations of other electron-induced

dissociationmethods [63] including electron-detachment dissociation (EDD) [64,65],

electron-ionization dissociation (EID) [66], and electron-transfer dissociation

(ETD) [67], which are collectively known as the ExD methods. In EDD, instead

of capturing a low-energy electron, a negatively charged precursor ion, when

bombarded by high-energy electrons, experiences loss of an electron from its valence

shell and forms a charge-reduced radical anion. This radical anionic species may

undergo backbone cleavages leading to the formation of a-, c-, and z-ions, much like

FIGURE 2.6 Amino acid preferences in �15,000 tandem mass spectra CAD and ECD.

Reproduced with permission from Elsevier (Zubarev R. A., et al. J. Am. Soc. Mass Spectrom.

2008, 19, 753). (See the color version of this figure in Color Plates section.)


those observed in ECD. EDD is particularly useful for fragmenting peptides that can

easily generate multiply charged anions in ESI; examples are phosphorylated,

sulfated peptides, or peptides with multiple acidic residues.

Given that both ECD and EDD are initiated by a charge-reduction process, they are

only applicable to multiply charged ions. Singly charged precursor ions, as those

produced byMALDI, would be neutralized by either electron capture in the positive-

ion mode, or electron detachment in the negative-ion mode, preventing the detection

of fragments. EID, however, can be applied to singly charged precursor ions.

The exact mechanism of EID is not well understood, but it probably involves first

the ionization of the precursor ion [M þ nH]nþ by interaction with a high-energy

electron to produce a radical [M þ nH](nþ 1)þ .

, which may dissociate directly or

capture a low-energy electron and then dissociate. EID spectra are often complex;

they show ions produced both by ergodic processes and by radical pathways.

In the early stages of development, ExD was used exclusively with FTICR

instruments; the presence of a magnetic field is beneficial for trapping thermal

electrons to allow efficient ion–electron interactions. More recently the implementa-

tion of ECDhas been successfully extended to other types ofmass spectrometers (e.g.,

a linear ion trap) with a superimposed magnetic field generated by a permanent

magnet [68].

ExD in anRF-only trap remained an elusive goal until 2004, when electron transfer

dissociation was developed. ETD takes advantage of ion–ion interactions, where

electron transfer from an anion radical, rather than the capture of an unbound electron,

initiates the bond dissociation. The reagent anions are generated in a negative

chemical ionization source (nCI), and introduced into the same ion trap where

positive peptide ions are stored. Commonly used anion reagents include aromatic

compounds with low electron affinities, such as azobenzene and fluoranthene, which

also have favorable Franck–Condon factors for transition from the ground vibronic

state of the anion to the low–lying vibrational states of the ground electronic state of

the neutralmolecule.An important competing reaction in ETDof peptides is a proton-

transfer reaction (PTR) that involves the movement of a proton from the multiply

protonated peptide precursor or fragment ion to the anion radical [69,70]. Although

PTR is often an undesired competition, it does have utility in ETD experiments

performed in low–resolving-power mass spectrometers, particularly for top–down

analysis of large protein ions. For the latter, PTR can reduce the charge state of the

highly charged fragment ions, enabling the accurate determination of their charge

states, and thus the mass values, which are otherwise difficult to obtain because

achieving isotopic resolution at higher charge states is difficult [71].

ETD shares many similarities with ECD, including the preferential and extensive

N�Ca bond cleavages, preservation of labile modifications, and ability to differenti-

ate certain isomeric amino-acid residues via secondary, radical-induced rearrange-

ments [72–74]. ETD is considered to give even “colder” fragmentation than ECD,

capable of retaining even sulfations, the most labile of PTMs. Its success stems from

the smaller amount of energy deposited than by ECD. One difference is that some

energy is needed to overcome the electron affinity of the anion reagent; another is the

collisional cooling afforded by a higher pressure ion trap. Like for ECD, the initially


formed c/z ion pair in ETD may still be held together by noncovalent interactions;

these need to be ruptured before the products can be detected individually. Overcom-

ing these noncovalent interactions is usually achieved by post-ETD collisional

activation, abbreviated as ETcaD [75]. ETD efficiency also increases with the charge

state of the precursor ion. ETD efficiency and sequence coveragemay be improved by

introducing fixed charge tags to the peptides, via, for example, amidation of the

carboxylic groups or derivatization of cysteines [76].

To date, there has been only one report of the ETD analog of EDD, where xenon

radical cations react with multiply deprotonated peptide anions to generate EDD-like

spectra for which a- and x-ions are the major fragments [77]. Current research efforts

using radical cations from polycyclic aromatic hydrocarbons (PAHs) as the reverse

ETD reagent (electron acceptor) for negatively charged peptide and carbohydrates

suggest promise for reverse ETD (rETD) in the structural analysis of biomolecules.

2.2.5 Other Radical-Induced Fragmentation Methods

ExD and UVPD are just two of several classes of fragmentation methods that involve

radical-induced reactions. Other methods that generate reactive radical peptide ions

include collisional activation and interaction with metastable atoms or other free

radicals. In free-radical-initiated peptide sequencing (FRIPS), a free-radical initiator is

conjugated to theN-terminus of a peptide; the initiator can be cleaved byCAD to leave

the radical on the peptide [78]. Subsequent collisional activation induces fragmenta-

tion of the peptide, generating abundant a- and z-type ions. Two advantages of FRIPS

over ExD are its ability to fragment singly charged peptide ions and the possibility of

generating radicals with different reactivities for selective gas-phase fragmentations.

In metastable-atom fragmentation (MAF) [79] or metastable-atom dissociation

(MAD) [80], the radical on the peptide is generated by collisions with metastable

atoms, usually electronically excited He*, Ne*, Ar*, or Kr*. Metastable atom beams

may be produced by electron impact, DC plasma discharge, or RF discharge. The

MAF process likely involves Penning ionization of the precursor ion by collisionwith

the metastable atom, generating a radical cation that undergoes ExD-like fragmenta-

tions. A typical MAF spectrum (Figure 2.7) of a peptide shows promise as an

alternative to ExD for odd-electron ion fragmentation, capable of producing extensive

backbone cleavages without the loss of labile PTMs. Further, MAF is not subject to

the charge-state limitation of ECD/ETD, and is applicable to singly charged and

negatively charged ions. The MAF source can also generate reactive radical species,

such as CH3. or OH., simply by doping the reagent rare gas with methane or water;

these radical species may abstract a hydrogen from the peptide precursor and initiate

fragmentation.

2.3 MASS ANALYZERS

TandemMS analysis usually requires selection of a precursor andmass analysis of the

products; both steps employ mass analysis. In this section we consider the principles

MASS ANALYZERS 59

of various mass analyzers that are used inMS/MS experiments in peptide and protein

MS. Mass analyzers determine the m/z value of a charged particle based on its

trajectory in electric or magnetic field or both. Different mass analyzers influence the

ion motions in different ways; some through the application of electrostatic or

electrodynamic fields, others via the use of a combination of electric and magnetic

fields. The fundamental law that governs the ion motion is Newton’s second law of

mechanics:

F ¼ md2r

dt2; ð2:3Þ

and the force an ion experienced in electromagnetic fields can be expressed as

F ¼ qEþ qv� B; ð2:4Þ

where the two terms on the right-hand side represent the electric and magnetic

components, respectively. Many different types of mass analyzers have been devel-

oped and applied to the structural analysis of proteins; the most common are the time

of flight, quadrupole, quadrupole ion trap, orbitrap, and Fourier-transform ion

cyclotron resonance mass spectrometers.

2.3.1 Time-of-Flight Mass Analyzer

A time-of-flight instrument [81] separates ions of different m/z based on their flight

times through a field-free drift region. TOF analyzers are well suited to pulsed ion

sources such asMALDI because they operate in a pulsed ion-countingmode. A linear

FIGURE 2.7 Fragmentation spectrum of substance P obtained via interaction with a low

kinetic energy beam of metastable argon atoms (* denotes contaminant peaks). Adapted with

permission from American Chemical Society (Berkout V. D., Anal. Chem. 2006, 78, 3055).


TOFanalyzer is conceptually the simplest type ofmass analyzer, whose principles are

illustrated in Figure 2.8. All ionswith the same number of charges z are accelerated by

an electrical potentialUs applied between the sample plate and an extraction electrode

to the same kinetic energy (Ek¼ zeUs), where e is the elementary charge, but with

different velocities v, as determined by

v ¼ffiffiffiffiffiffiffiffiffiffiffiffi2zeUs

m

r: ð2:5Þ

The flight time of an ion through the drift region is given by

t ¼ Lffiffiffiffiffiffiffiffiffiffi2eUs

p m

z

� �1=2

: ð2:6Þ

After exiting the flight tube, the ions strike a detector successively in the order of

theirm/z values, from low to high. Them/z value of a given ion arriving at time t can be

calculated by using the calibration equation

m

z

� �1=2

¼ A*tþB; ð2:7Þ

whereA is determined by the flight tube length and the acceleration voltage, andB is a

correction term for time zero offset, which may be caused by trigger delay or

propagation delay in the detector circuit.

A simple detector for TOF instruments is a secondary emission multiplier (SEM)

consisting of a series of dynodes held at decreasing negative potentials. Ions striking

the surface of the first dynode cause an emission of secondary electrons, which are

then accelerated toward the next dynode held at a less negative potential, generating

more secondary electrons upon impact. This process may continue as the electrons

travel toward the ground potential, leading to a cascade of electrons. The final electron

flow out of the last dynode will be orders of magnitude higher than the initial one

emitted from the first dynode; the amplified current can be converted to a voltage that

is easily detected by a conventional electronic amplifier.

Alternatively, a microchannel plate (MCP) may be employed as the detector. An

MCP is essentially a glass plate with many channels, whose surfaces are coated to

FIGURE 2.8 Principles of a linear time-of-flight mass analyzer.

MASS ANALYZERS 61

achieve a high ion/electron conversion and electron-multiplication yield. The inner

surface of each channel resembles a continuous array of dynodes; the potentials vary

from high to low negative values from near the front to the back surface of theMCP, as

sustained by applying an around 1 kV voltage difference between the two sides of the

plate. Ions hitting the front surface of the MCP will induce emission of electrons that

cascade down the channel, much like what happens in the SEM. Compared to SEMs,

MCPs have the advantage of a faster response time, but they can be easily saturated

because their recovery time is long.

The analog electron current signal can be digitized by using either a standard

analog-to-digital converter (ADC) or a time-to-digital converter (TDC). An ADC

samples the analog detector voltage at discrete intervals and stores the digitized value

in a memory from which the signal can be reconstructed or read out by the computer.

ADCs for modern TOF instruments can operate at a sampling rate of 1–4GHz, but

only with an 8-bit board, which limits the dynamic range of stored signal amplitudes

to a maximum of 256. Moreover, saturation may occur in both the digitizer and the

upstream analog current-to-voltage amplifier, leading to flat-topped peaks and

erroneous ion-abundance measurements. ATDC is like a 1-bit ADC, which records

the arrival time of ions not as an analog signal, but as an array of 1’s and 0’s. The ion

abundance information is recovered by summing over a large number of spectra. A

TDC has the advantage of ultra-fast response and data-transfer rate. ATDC, however,

suffers from its unit dynamic range; it is particularly undesirable when multiple ions

strike the detector at the same time,which results inmissing the signal from the slower

arriving ions. Further, these detectors suffer from dead-time issues, which occurs

when one ion strikes the detector so closely following another that the detector cannot

respond to the second ion. Generally, TDCs are used in orthogonal TOF instruments,

whereas the ion-extraction optics can operate at a very fast repetition rate (several

kHz), and each extracted ion packet contains only a small number of ions.

The mass resolving power of a TOF instrument scales with the length of the flight

tube. There is usually a practical limit on how long a flight tube can be, and a longer

flight tube is also associated with decreased sensitivity caused by ion loss due to

angular dispersion of the ion beam. TOF mass resolving power is also limited by the

timewidth of the ion packet arriving at the detector, which is determined by variations

in when and where they are formed, as well as their kinetic-energy spread.

The TOFbroadening caused by the kinetic energy spread can be partially corrected

by using a delayed extraction (DE) scheme [82–84], as shown in Figure 2.9. With

continuous extraction, all ions are extracted and accelerated by the same electric

potentialUs, and the ionswith a higher initial velocitywill arrive at the detector earlier

than the ions (with the same m/z) with a lower initial velocity (Figure 2.9A). With

delayed extraction, ion extraction and acceleration are done in two stages

(Figure 2.9B). The extraction potential Ue is not applied until after a certain delay

time following the ion formation, during which time the ion packet will expand in

space. Ions with lower initial forward velocity will not move as far down the

extraction field as those with higher initial forward velocity, and consequently will

experience more acceleration when the extraction voltage is turned on, allowing

them to catch upwith the fastermoving ions. Careful selection of the delay aswell as


the extraction and acceleration voltages, Ua, permits all ions of a specificm/z value

to be “time focused” to arrive at the detector at nearly the same time regardless of

their initial velocities. It should be noted that the DE focusing condition is mass

dependent, and the resulting improvement in mass resolving power drops signifi-

cantly when applied to a broad m/z range.

TOF broadening resulting from the ion kinetic energy spread may also be

reduced by employing an electrostatic ion mirror, known as the reflectron [85],

as shown in Figure 2.10. The reflectron is usually a stack of ring electrodes, located

at the end of the flight tube; when the electrodes are “turned on,” they create a

constant electric field, usually through a linear voltage gradient, that slows down the

ions and turns them around toward the detector located at the other end of the flight

tube. For ions of a given m/z value, those with higher initial kinetic energy will

penetrate more deeply the reflectron field, spending more turn-around time inside

the reflectron, thus partially compensating their shorter flight time outside of the

reflectron. With proper setting of the reflectron voltage, Ur, ions with both high and

low initial kinetic energies can be focused at the detector. The best reflectron-

focusing condition is typically achieved when the ion spends an equal amount of

time inside and outside of the reflectron. Modern TOF instruments equipped with

both DE and a reflectron can routinely achieve mass resolving powers of 420,000

and mass accuracies in the 2 to 5-ppm range.

FIGURE 2.9 Schematic of a linear TOF instrument with (A) continuous ion extraction and

(B) delayed pulsed ion extraction showing that TOF broadening caused by the kinetic energy

spread of ions can be reduced by employing a delayed ion extraction scheme.

MASS ANALYZERS 63

TOF mass resolving power may also be improved even more by adopting an

orthogonal TOF (oTOF) arrangement [86,87], a schematic of which is shown in

Figure 2.11. In an oTOF instrument, instead of being accelerated along its axis of

motion, the ion packet is extracted and accelerated sideways by a pulsed voltage

applied to the deflector. Because the resulting TOFaxis is perpendicular to the original

axis of ion motion, the initial ion kinetic energy spread will not signficantly

compromise the achievable mass resolving power. oTOF instruments often include

a high-pressure quadrupole for ion cooling and focusing, as well as additional ion

optics to squeeze the ion packet both radially and axially for improvedmass resolving

FIGURE2.10 Schematic of a reflectronTOF instrument illustrating the focusing effect of the

reflectron on ions of the same m/z but with different initial kinetic energy.

FIGURE 2.11 Schematic of an orthogonal TOF instrument.


power. A distinct advantage of oTOF instruments is that they can work with

continuous ion sources, such as ESI because the pulsed TOF analyzer is decoupled

from the ion source.

Tandem MS in TOF Instruments Tandem MS experiments cannot be per-

formed on a simple linear TOF mass spectrometer because the ion velocity is already

established as the ion enters the field-free flight tube; thus a fragment ion formed in the

drift region, via postsource decay (PSD), will have the same velocity as its precursor

ion, hence the sameTOF.On the other hand, PSD fragments can bemass analyzed on a

reflectron TOF instrument because they have kinetic energies that are proportional to

theirm/z [88]. This change in kinetic energy comes about because the kinetic energy

of the precursor must be conserved; thus it is partitioned between the fragments as a

function of their masses. Despite having the same initial velocity as the precursor ion

and the same flight time outside of the reflectron, a PSD fragment with its lower

kinetic energy will not penetrate the reflectron as deeply as the precursor ion. Lighter

fragments will spend less time inside the reflectron, and arrive at the detector earlier. It

is important to note that the focusing condition for each PSD fragment is different, and

a complete PSD spectrum generally requires piecing together multiple spectra

obtained at several different reflectron voltages, each covering only a fraction of

the mass range. Precursor ion selection is usually achieved by placing a pair of

electrodes outside of the source region to deflect unwanted ions, although aBradbury–

Nielsen gate, consisting of a set of alternatively biased wires with voltages applied at

high frequency, which allows ion passing only in certain voltage phase, is sometimes

used. These PSD spectra do not have high mass resolving power and are not

extensively used today in peptide sequencing.

Tandem MS experiments can also be performed more effectively on a TOF/TOF

instrument, which consists of two TOF analyzers in tandem, with a typical configu-

ration as shown in Figure 2.12 [15]. The first TOF analyzer is usually a short linear

drift tube, separating ions according to theirm/z values. A timed ion selector in front

of the collision cell is switched open at a proper delay to select a small m/z range

including those precursor ions of interest for CAD. The collisional energy can be

adjusted by changing the offset potential of the collision cell. The fragment ions

formed can be re-accelerated into the second TOF region, typically a high–resolving

FIGURE 2.12 Schematic of a tandem TOF instrument.

MASS ANALYZERS 65

power reflectron for mass analysis. ATOF/TOF mass spectrometer is one of the only

two instruments (the other one being tandem sector instruments, which are not widely

used for protein analysis) that are used for conducting high-energy CAD experiments

(high-energy activation can also be carried out with an FTICR instrument, but

the efficiency of product-ion detection is poor). UVPD can also be implemented in

TOF/TOF instruments, as the pulsed laser can both time-select and optically excite the

precursor ions [26].

2.3.2 Quadrupole Mass Analyzer and Quadrupole Ion Trap

Quadrupole Mass Analyzer A quadrupole mass analyzer, or quadrupole mass

filter (QMF), separates ions of differentm/z based on the stability of their trajectories

inside an RF field [89]. An ideal quadrupole contains four highly parallel metal rods,

of hyperbolic cross section, arranged in a square configuration. Each pair of the

opposing rods is connected electrically. AnRF voltage is applied between two pairs of

rods, with a DC voltage superimposed on it, creating a quadrupolar field inside the rod

arrangement; this field can be expressed as

fx;y ¼ðUþVcosWtÞ

r20ðx2�y2ÞþC; ð2:8Þ

where U is the DC voltage, V is the RF amplitude, W is the RF frequency, r0 is the

radius of the circle inscribed in the inner surface of the quadrupole, x and y are the

cartesian coordinate positions of the ions, and C is a constant voltage offset. In

practice, most quadrupoles use circular rods because they are easier to construct than

hyperbolic electrodes.With proper choice of the rod diameter and inter-rod distance,

a cylindrical quadrupole can produce an electric field that closely approximates a

quadrupolar field (see Figure 2.13 for the geometry of a quadrupole constructed with

circular rods).

FIGURE 2.13 Schematic of a quadrupole mass analyzer with cylindrical rods.


The ion motion inside a quadrupolar field can be described by the following

equations:

md2x

dt2¼ �e

df

dx¼�2ze

ðUþVcosWtÞr20

x ð2:9aÞ

and

md2y

dt2¼�e

df

dy¼ 2ze

ðUþVcosWtÞr20

y; ð2:9bÞ

where z is the number of charges the ion carries, rather than the cartesian coordinate.

Equations (2.9) can be rewritten in the more familiar form of theMathieu equation

by expressing the instrumental parameters as four dimensionless numbers:

ax ¼�ay ¼ 8zeU

mr20W2

ð2:10aÞ

and

qx ¼�qy ¼ �4zeV

mr20W2: ð2:10bÞ

The stability of the ion trajectory depends on the ion’s a and q values. A stable ion

trajectory is one where the ion motion is bound in both x- and y-dimensions (i.e.,

|x|5 r0 and |y|5 r0 at all times). Although many stability regions exist in the a/q

space, nearly all commercial quadrupoles operate in the first stability region, as

outlined in Figure 2.14. It is evident from equations (2.10) that the stability diagram in

the a/q space can be directly translated into the U/V space with a scaling factor that is

proportional to the ion’s mass-to-charge ratio. Figure 2.15 depicts the stability

diagrams of several ions of different m/z values. Normally, when the quadrupole is

used as a mass analyzer, the DC potential and the RF amplitude are ramped together

while the ratio ofU/V is kept constant, along the operating line as shown in Figure 2.15

(dashed line). Theoperating line scans across the tips of the stability region of eachm/z,

allowing the sequential passage of ions of differentm/z values, from low to high. Near

the vertex of the stability diagram of a givenm/z ion, only ions within a small window

of that m/z can have stable trajectories and be transmitted. Given that it takes a finite

time for an ion to “fly” through the quadrupole, theU andV valuesmust be kept within

its stability region for thewhole length of the ion residence time inside the quadrupole,

limiting the scan speed. The scan speed can be increased by decreasing the slope of the

operating line, thus increasing them/zwindow of transmission at any given instant. A

broader transmission window, however, also means a reduced mass resolving power.

Modern quadrupolemass analyzers can achieve amass resolving power of up to nearly

10,000 in a high–resolving powermode, and a scan speedwell over 10,000 u/sec at unit

mass resolving power (i.e., sufficient to separate adjoiningm/z ions). The upper mass

limit of commercial quadrupoles is typically between m/z 4000 and 6000, which is

limited by the maximum amplitude the RF power supply can provide, and by its

frequency, which is normally a constant and cannot be readily varied by the operator.

MASS ANALYZERS 67

As suggested by the stability diagram, quadrupoles can be used as ion guides when

operated in the RF-only mode. With no DC potential applied (a¼ 0), all ions above a

certain m/z values can be transmitted. This low mass cutoff may be decreased by

applying lower RF amplitude, V, allowing, in principle, ion transmission across a

FIGURE 2.14 Stability diagram of a quadrupole in a/q space.

FIGURE 2.15 Principles of a quadrupole mass analyzer.


wider mass range. In practice, however, the ion transmission efficiency is also limited

by the focusing ability of the quadrupole. Ion dispersion in an angular way toward the

quadrupole rods may be caused by a number of reasons, including the initial ion

velocity, which often contains a radial component, space-charge effects, which

originate from the mutual repulsion of ions of like charge within the ion packet,

and scattering collisions with residual or background gas molecules. The focusing

ability of the quadrupole is characterized by the depth of the effective radial trapping

potential well, which is proportional to V2. Ions with a higher m/z value are less

affected by the electric field, requiring a deeper trapping well to be efficiently

focused and transferred. When transferring ions across a broad mass range, there is

often a compromise in choosing the RF amplitude to avoid the loss of low-mass ions,

owing to their unstable trajectories, and to reduce the loss of high mass ions, owing

to inefficient focusing.

Tandem MS in Triple-Quadrupole Mass Spectrometers Tandem MS

experiments cannot be performed on a stand-alone quadrupolemass analyzer because

it lacks ion-trapping capability. Spectrometers employing multiple quadrupoles in

series, on the other hand, are widely used for tandemMS analysis [90–92]. The most

common type is the triple quadrupole instrument, as shown in Figure 2.16. The first

and third quadrupoles (Q1 andQ3) are used asmass filters, and the second quadrupole

(q2) is operated in the RF-only mode and used as a collision cell.

Given thatQ1 andQ3 can be either tuned to a fixedDC/RFvalue formass selection,

or scanned to perform mass analysis, a triple quadrupole mass spectrometer can be

operated under four different modes. The most commonly used mode is the product-

ion scan, whereQ1 is tuned to select precursor ions of a specificm/z, which are further

subjected to CAD in q2. Q3 is scanned to measure the fragment ion masses. The

product-ion scan is frequently used in liquid chromatography LC-MS/MS, and is very

useful for deducing the structure of precursor ions. Alternatively, Q1 can be scanned,

while Q3 is held at a constant DC/RF value to allow detection of a particular fragment

ion of interest. This precursor-ion scan mode is particularly useful for identifying all

precursor ions that produce a common product ion of interest. The third scanning

mode is the neutral loss scan, where Q3 is scanned at the same rate as Q1, but with an

offset, Dm. The value of Dm is usually negative, and the resulting mass spectrum

displays all precursor ions that undergo the same neutral loss upon collisional

FIGURE 2.16 Schematic of a triple quadrupole mass spectrometer.

MASS ANALYZERS 69

activation in q2. The neutral loss scan is often used to screen for a class of compounds

that contain a similar labile group (e.g., a phosphate group that is eliminated as neutral

phosphoric acid from phosphorylated peptides). The final mode is the selected

reaction monitoring (SRM), sometimes also referred to as the multiple reaction

monitoring (MRM)mode, where Q1 and Q3 are both operated at fixedDC/RF values,

with Q1 allowing passage of a particular precursor ion and Q3 allowing transmission

of an expected fragment ion from the selected precursor [93]. SRM is usually

performed when the instrument is coupled with LC, producing a chromatogram of

a specific precursor ion corresponding to a specific analyte of interest. Because both

mass analyzers are operated at fixedm/z values, SRM is a highly specific and sensitive

method for identifying compounds, provided that their fragmentation behaviors are

known.

Linear Quadrupole Ion Trap A quadrupole can be turned into an ion-storage

device by adding end trapping electrodes or by segmenting the quadrupole (see

Figure 2.17 for a linear quadrupole ion trap (LIT) with sectioned rods) [94,95]. The

segmented quadrupole has the advantage ofminimizing potential ion losses caused by

the fringe field near the end of the center quadrupolewhen themainRF voltage used to

confine the radial ion motion is also applied to the end quadrupole segments. A DC

potential is applied to the end segments to confine the axial ion motion, and collision

gas is also used to facilitate the ion trapping. Two of the four center quadrupole rods

(the X-pair in the diagram) have open slits in the middle for radial ion ejection during

mass analysis. A conversion dynodewith an electronmultiplier is placed on each side

of the ion trap to allow detection of most ejected ions. Compared with the 3D

quadrupole ion trap (discussed below), a linear ion trap has the advantage of a higher

space-charge limit as it allows ion clouds to expand axially over a larger volume.

FIGURE 2.17 Schematic of a linear quadrupole ion trap.


Unlike a quadrupole mass analyzer which uses a combination of RF and DC

voltages to achieve mass selection, an LIT performsmass analysis by scanning the RF

amplitude alone, without anyDC potential applied between the two sets of quadrupole

rods. As evident from the stability diagram of a quadrupole trap, Figure 2.14, only ions

with q5 0.908 have stable trajectories along the q-axis (where a¼ 0). Recall that q is

proportional to V/(m/z); as the RF amplitude is ramped up, lowm/z ions will reach the

edge of the stability region first, and they are ejected from the trap. Although

conceptually simple and easy to implement, such mass-selective instability scan has

several drawbacks, including slow ion ejection near the edge of the stability region and

low detection efficiency because only a portion of the ions actually exit from the slits.

An improved way to perform mass analysis using an LIT is through resonance

ejection. Each ion has its unique oscillation frequency inside a quadrupolar field,

known as the secular frequency fs, which differs from the fundamental RF frequency.

Because heavier ions have slower response to the change of the electric field, they

oscillate at lower frequencies than those of lighter ions. The secular frequency is

related to the ion q value (defined in equation 2.10), and fs increases as q increases.

During the mass analysis, a small auxiliary AC voltage at a fixed frequency is applied

between the two X-rods only. As the main RF amplitude is ramped up, all ions

experience an increase in their q values and consequently their secular frequencies.

When the secular frequency of a certain ion reaches the frequency of the auxiliary AC,

it will be resonantly ejected from the trap and detected. The AC frequency chosen

allows ions to reach resonance at a q value slightly below 0.908, which permits faster

ejection than allowing the ions reach the edge of the stability region; the outcome is

improved mass resolving power. Furthermore, the sensitivity is also improved via

resonance ejection because ions exit the trap almost exclusively along the x-axis, with

minimal scattering loss.

Given that an LIT is an ion-trapping device, tandemMS analysis is possible using a

single LIT. Precursor ion selection is achieved by applying a tailored RF waveform to

the X-rods, which contains RF power at the secular frequencies of all ions except for

the ion of interest. Selected ions are then activated by applying a resonant dipolar

excitation waveform along the X-rods, with the amplitude kept low to avoid ion

ejection. Collision energy can be increased by performing resonance excitation at a

higher q. A higher q, however,means a smallerm/z range for detectable fragment ions.

For example, for a precursor ion of around m/z 908 excited at around q 0.30, all

fragment ions with m/z less than 300 will have a q value above 0.908, and thus be

unstable and undetectable. A pulsed Q dissociation (PQD) scheme can be imple-

mented to alleviate this problem. In PQD, precursor ions are first resonantly excited at

a higher q value and held there for a short period of time for collisional excitation, but

not long enough for significant dissociation to occur.After this short excitation period,

the main RF amplitude is dropped to bring the q values down before or as the ions

dissociate, and the fragment ions are trapped at low q values.

Alternatively, photodissociation, either UVPD or IRMPD, or ETD can be used to

circumvent the compromise between ion activation and fragment-ion trapping. The

geometry of an LITallows easy axial introduction of the laser beam, and the extended

ion-storage time permits extensive ion–photon interactions for efficient precursor-ion

MASS ANALYZERS 71

excitation [24]. The elevated pressure in an LIT is sometimes undesirable for IRMPD

as the rapid collisional cooling can compete with ion activation. For ETD, simulta-

neous axial trapping of analyte cations and ETD reagent anions is achieved by

applying an RF trapping voltage to the end lenses of the linear trap.

3D Quadrupole Ion Trap A 3D quadrupole ion trap (QIT), or Paul trap, works

under the same principle that governs the operation of an LIT [96–98]. AQIT consists

of a cylindrical ring electrode and two end caps. Conceptually a QIT can be

considered as a quadrupole that bends around itself to form a closed loop. As the

inner radius of the resulting “donut” shrinks, the inner rod is reduced to a point,

the outer rod becomes the ring electrode, and the top and bottom rods become the end

caps (see Figure 2.18 for a schematic). Externally generated ions enter the trap

through an opening in one end cap, and are trapped by a combination of the alternating

electric field and collisions with buffer gas that refocus the ions to the center of the

trap. Ions are ejected through an opening on the other end cap during mass analysis

and ion detection.

Ion motion inside the QIT is also described by the Mathieu equation, although the

expressions of a and q are slightly different from those for the linear quadrupole ion

trap because they have different geometries:

az ¼�2ar ¼ �16zeU

mðr20 þ 2z20ÞW2ð2:11aÞ

and

qz ¼�2qr ¼ 8zeV

mðr20 þ 2z20ÞW2: ð2:11bÞ

Like its 2D cousin, a 3D QIT works along the q-axis, with no DC potential

difference applied between the ring electrode and end caps. Most of the time the

main RF is only applied to the ring electrode, while the end caps are primarily used

FIGURE 2.18 Schematic cross-sectional view of a 3D quadrupole ion trap.


for resonant-ion ejection and excitation. Mass analysis can be performed either by

mass-selective instability scan or by resonance ejection. The upper mass limit is once

again limited by the ability of the RF power supply to provide a sufficiently high

voltage to drive the qz over 0.908. Although a reduction in trap dimension can lead to

an increase in its upper mass limit, it is also associated with reduced ion-storage

capacity due to coulomb repulsion between like charges. Alternatively, the upper

mass limit can be increased by performing resonance ejection at a frequency

corresponding to a lower q.

CAD experiments can be similarly carried out on a 3D QIT, but loss of low mass

fragment ions can occur as the V is increased for precursor-ion activation. Alternative

fragmentationmethods employing ion/ion interactionsmay benefit because a 3DQIT

is a charge-sign-independent trapping device, which makes it particularly suitable for

ETD tandem MS analysis. Consecutive tandem MS analysis (MSn) can be easily

performed on aQIT, by alternating the ion-selection and ion-fragmentation steps. The

low cost, compact size, rapid analysis time, andMSn capability of aQITmake it one of

the most common instruments for LC/MS-MS analysis.

2.3.3 Fourier-Transform Ion Cyclotron Resonance Mass Spectrometer

The Fourier-transform ion cyclotron resonance (FTICR) MS was developed in the

1970s by Comisarow and Marshall [99,100]. An FTICR mass spectrometer deter-

mines the m/z value of ions based on their cyclotron frequencies in a homogeneous

magnetic field. The Lorentz force an ion experienced in a magnetic field of strength B

is normal to its velocity v and the magnetic field lines, causing the ion to undergo

cyclotron motion, with the Lorentz force balancing the centrifugal force:

F ¼ zevB ¼ mv2

r: ð2:12Þ

Thus the unperturbed angular cyclotron frequencyvc of an ion of a givenm/z value

in a fixed magnetic field is given by

vc ¼ v

r¼ Be

m=zð2:13Þ

where e is the elemental charge, and z is the charge in integers.

In practice, because a homogeneous magnetic field can only confine the ion

motion in the radial direction (i.e., the direction perpendicular to the magnetic field

line), an inhomogeneous electrostatic field is also applied to trap ions in the axial

direction. The axial electrical trapping results in an axial oscillation with the

frequency vz, given by

vz ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2zVtrapa

ma2

r; ð2:14Þ

where Vtrap is the trapping potential applied, a is the dimension of the ICR trap, and

a is a constant that depends on the geometry of the trap. The electric field and the

MASS ANALYZERS 73

resulting axial harmonic motion reduce the cyclotron frequency and introduce a

second radial motion called the magnetron motion. The natural angular frequencies

of the ion motions are now

v� ¼ vc

2�

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffivc

2

� �2

� vz

2

� �2r

; ð2:15Þ

where vþ is the reduced cyclotron frequency and v� is the magnetron frequency.

It is this reduced cyclotron frequency vþ that is measured in FTICR. Thus a proper

calibration equation is needed to correct for the trapping term when calculating the

m/z value of an ion based on its measured reduced cyclotron frequency [101].

The simplest ICR trap is of cubic shape, which consists of three pairs of metal

plates orthogonal to each other (Figure 2.19). The pair of plates that is perpendicular to

the magnetic field is used as trapping plates, to which a small DC voltage is applied to

confine the ion motions along the z-axis. The other two pairs of plates are used as

excitation and detection plates, respectively. Although this is an informative trap for

explaining the principles, most modern traps are cylindrical in design.

Figure 2.20 illustrates the principle of operation for an FTICR mass spectrometer.

The initially trapped ions are confined radially to very small cyclotron radii owing to

their thermal velocities. For example, at room temperature a singly charged ion of

m¼ 100Da in a magnetic field of 12 T has a thermal ICR orbital radius of around

0.02mm. This small-amplitude thermal cyclotron motion is not useful for ion

detection because it is neither coherent nor can it induce significant image currents

on the detection plates. The ion packet may be excited to a larger orbit by applying an

azimuthal (i.e., perpendicular to the magnetic field) spatially uniform field that is

FIGURE 2.19 Schematic of a cubic FTICR trap.


oscillating sinusoidally with the same angular frequency, vc, as the ion’s character-

istic angular cyclotron frequency. After the excitation all ions of the same m/z value

will move coherently as a tight packet in a larger cyclotron orbit, inducing alternating

image charges on the opposing detection plates. The induced alternating image

current has the same frequency as the ion cyclotron frequency.Multiple ion packets of

differentm/z values can be excited to the same cyclotron radius, albeit with different

frequencies, by applying a swept rf excitation waveform with equal magnitude for all

frequencies (also known as the “chirp” excitation). Image current induced by all ion

packets can be detected simultaneously as a superposition of many sinewaves, which

are amplified, digitized, and stored as a time-domain transient. This transient

is Fourier-transformed to give a frequency-domain spectrum, and finally mass-

calibrated to produce the mass spectrum.

The Fourier-transform limitedmass resolving power of an FTICRmass analyzer is

roughly equal to f*t/2, where f is the cyclotron frequency and t is the transient length.

Thus, it is very important to maintain an ultra-high vacuum (typically�10�10 torr) in

the ICR region; otherwise, collisions of ions with background gas may lead to rapid

transient decay and poor mass resolving power. One may calculate that an FTICR

mass spectrometer with a 7 T magnet can provide around 100,000 resolving power at

m/z 500 with a one-second transient. The high resolving power may also be

appreciated by considering the distance an excited ion traverses in its orbital motion

in a short time. In the example above, if the ion were excited to an orbit of 5 cm in

radius, it would travel a distance of around 63 km during a one-second observation

FIGURE 2.20 Principles of FTICR mass spectrometry.

MASS ANALYZERS 75

time. Given that the frequencies can be measured with high accuracy, their corre-

spondingm/z can also be calculated with high accuracy.With a well-constructed ICR

trap and careful control of experimental conditions, a modern FTICR mass analyzer

can routinely achieve mass accuracy of52 ppm with external mass calibration, and

into the ppb region with internal calibration.

TandemMS in FTICRMass Spectrometers Because an FTICR trap can store

ions, tandemMSanalysis can be easily performedwith an FTICRmass spectrometer.

A precursor ion may be isolated by applying tailored excitation waveforms, such as

the SWIFT (stored waveform inverse Fourier transform) [102]. Precise ion isolation

down to a 0.1Da m/z window as well as multiple precursor-ion selection can be

achievedwith SWIFT. The ICR trap is particularlywell suited for performing several

tandem MS experiments, including IRMPD and ECD. IRMPD in an ICR trap

benefits from its long ion-storage time, which allows extensive ion–photon interac-

tion, and its ultra-high vacuum, which minimizes collisional cooling. Until very

recently an FTICR instrument is the only type ofmass spectrometer that is capable of

performing ECD analysis, primarily because of its ability to guide and trap electrons

with magnetic field.

CAD can also be used to fragment ions in an FTICR mass spectrometer, but

generally not with resonant excitation because ions can be lost owing to the high-

energy collisions, and collisional damping is required if one to bring the product ions

back to the center of the trap for re-excitation for mass analysis. With resonant

excitation, fragment ions tend to be formed off-axis and, given large magnetron

motion amplitudes, produce poor spectra and extensive fragment-ion losses. Instead,

selected ions are usually excited by a slightly off-resonance waveform that periodi-

cally excites and de-excites the ions, ensuring ample collisions while keeping the ions

relatively close to the center of the ICR trap [9]. Although such sustained off-

resonance irradiation (SORI) can produce product-ion spectra similar to those

obtained by other low energy CAD methods, direct introduction of the collision gas

into the ICR trap is undesirable, as a long pump-down delay is usually needed after the

fragmentation event to achieve the low pressure that is suitable for high-resolving-

power mass analysis. The extra delay leads to low duty cycles, and thus SORI-CAD is

not suitable for high-throughput tandem MS analysis, particularly when the mass

spectrometer is coupled with LC.

Nearly all modern commercial FTICR instruments are hybrid instruments that

employ either a linear ion trap or a QMF-collision cell setup as the front end. Given

that CAD performed in the front end does not compromise the vacuum in the ICR

trap, these hybrid instruments are ideal for performing high-throughput, high-

mass-accuracy LC-MS/MS analyses. In addition, the front end can also be used to

isolate ions, without inducing significant magnetron motions. The LIT offers the

possibility of automatic gain control (AGC) for maintaining constant ion popula-

tions in the ICR trap, which is crucial for achieving high mass accuracies. The

QMF-collision cell setup, on the other hand, allows selected ion accumulation,

which is beneficial for analysis of low-abundance ions, leading to dramatically

increased dynamic range.


The excellent mass resolving power and accuracy achievable on an FTICR and its

versatile tandem MS analysis capability make FTICR optimal for many applications

(e.g., top-down proteomics [103] where whole intact protein ions are fragmented in

the gas phase for identification and characterization, and complex mixture analysis in

petroleomics research [104]). The analysis time on FTICR mass spectrometers,

however, is long, relative to times of chromatographic separation, with a typical

acquisition time around one second to achieve a reasonably high mass resolving

power. In addition, FTICR instruments are usually expensive, owing to the cost for the

magnet, further limiting their application in routine sample analysis.

2.3.4 Orbitrap

Ion trapping by a pure electrostatic field is also possible. The first electrostatic ion trap

was developed byKingdon in the early 1920s; it consists of a central wire and an outer

cylindrical electrode to produce a radial electrostatic trapping field. An ion circles

around the central wire (orbital motion) in a Kingdon trap, with the centrifugal force

balanced by an attractive coloumbic force. The outer electrode was later modified to

include an axial quadrupolar term for axial ion-motion confinement. Neither config-

uration was reported to produce mass spectra. The breakthrough came in the late

1990s with the development of the orbitrap by Makarov. Aside from redesigning the

electrodes to generate a quadro-logarithmic electric field,Makarovalso devised away

to introduce externally generated ions into the orbitrap, and more important, with the

same initial phase with regard to their axial motion [105–107].

The schematic cross section view of an orbitrap (Figure 2.21) shows a trap

consisting of an inner spindle-shaped electrode and an outer barrel-shaped electrode,

FIGURE 2.21 Schematic cross-section view of an orbitrap mass spectrometer.

MASS ANALYZERS 77

which is sectioned in the middle. The electrostatic field inside the orbitrap can be

described by a quadro-logarithmic distribution:

Uðr; zÞ ¼ k

2z2� r2

2

� �þ k

2R2mln

r

Rm

� �þC; ð2:16Þ

where z and r are cylindrical coordinates, with z ¼ 0 being the plane that bisects the

outer electrode,k is the field curvature,Rm is the characteristic radius of the trap, andC

is a constant voltage offset. Stable ion trajectories inside the orbitrap combine orbital

rotations around the inner electrode and harmonic oscillations along it. Several m/z-

dependent characteristic frequencies exist, including the frequency of rotationvf, the

frequency of radial oscillationvr, and the frequency of axial oscillationvz. Recall that

the restoring force along the z-axis can be calculated as

Fz ¼�qqUqz

¼�qkz ¼ md2zdt2

; ð2:17Þ

which describes a simple harmonic oscillator, with the frequency of oscillation being

vz ¼ffiffiffiffiffiffiffiffiffik

m=q

s¼

ffiffiffiffiffiffiffiffiffike

m=z

s; ð2:18Þ

where e is the elemental charge.

Ion oscillation along the z-axis induces an image current between the two parts of

the sectioned outer electrode. As for FTICR MS, this image current can be Fourier-

transformed to generate the frequency domain spectrum, and further mass-calibrated

to produce the mass spectrum.

Successful generation of mass spectra by using an orbitrap hinges on the ability to

introduce the externally generated ions as a tight packet into the orbitrap, so that the

axial motion of ions of the samem/z is coherent. The original orbitrap design adopted

a high voltage pulsed ion deflector to achieve this purpose, although only a small

fraction of the ions made their way into the orbitrap. The latest commercial orbitrap,

the LTQ-Orbitrap, employs a C-trap, which is a curved quadrupole that can be pulsed

to push all trapped ions into the orbitrap, all with the same initial phase axial motion

(Figure 2.22) [108,109].

As for all Fourier-transform mass analyzers, the Fourier-transform limited mass

resolving power of an orbitrap is approximately equal to the product of the measured

frequency (in this case the frequency of ion oscillation along the z-axis) and the

transient length. The commercial orbitrap can routinely achieve a mass resolving

power of around 100,000 atm/z 400 for a 1.25 s transient. This mass resolving power

is similar to that obtainable on an FTICR mass analyzer with a 4.3 T magnet at the

samem/z and with the same transient length. Because the z-axis oscillation frequencyof an ion inside an orbitrap has a weaker dependence onm/z (equation 2.18) than the

cyclotron frequency of an ion inside an ICR trap (equation 2.13), the mass resolving

power of an orbitrap does not decrease as fast as that of an FTICR as them/z increases,

and can exceed that of a commercial FTICRwith a higher field (7 or 9.4 T)magnet for


ions of higher m/z for the same acquisition time. The longest attainable transient

produced by an orbitrap (currently at approximately 2 s), however, is significantly

shorter than that by an FTICR (4100 s); thus the ultimate mass resolving power

achievable on an orbitrap is also significantly lower. Transient decay in an orbitrap

results from ion loss and dephasing, which are caused by several factors including ion

collisions with background gas, field imperfections, instability of power supplies, and

space-charge effects.

The LTQ-Orbitrap can achieve amass accuracy in the low ppm rangewith external

calibration. A “lock-mass” standard, such as polycyclodimethylsiloxane (PCM-6)

ions (m/z 445.1200) generated as the background ions during the ESI process from a

siloxane contaminant in the atmosphere, can be added to the analyte ion packet via

sequential filling of the C-trap. The presence of the lock-mass ion in the same ion

population as the analyte of interest provides an internal standard formass calibration,

allowingmassmeasurement with better than 1-ppmmass accuracy. Scan speeds of up

to 5 scans/s are possible, albeit with reduced mass resolving power and mass

measurement accuracy.

TandemMS in LTQ-Orbitrap An ion packet of a specificm/z in the orbitrap can

be selectively excited or de-excited by applying a resonant dipolar AC signal to each

half of the outer electrodes. Although such ability to manipulate confined ion

populations allows precursor-ion selection and excitation, tandemMS analysis inside

the orbitrap has not been demonstrated to date. In a commercial orbitrap, ion

fragmentation is typically done in the front end linear ion trap (called an LTQ in

the commercial instrument). The LTQ can be used to perform low-energy CAD and

ETD analyses, as well as a full range ofMSn experiments. Fragment ions can be mass

analyzed in the LTQ for high-throughput analysis, or in the orbitrap if high mass

resolving power and accuracy are desired. For ETD experiments, reagent anions can

LTQ transfer octopole C-trap collisionoctopole

HCD

Orbitrap

FIGURE 2.22 Schematic of the LTQ-Orbitrap XL instrument. Adapted with permission

from Macmillan Publishers Ltd: [Nature Methods] (reference [108]), copyright (2007).

MASS ANALYZERS 79

be introduced into the LTQ either from the front or from the back. The original front-

loading design employs a dual, pulsed nanoESI (nESI) source, with the second nESI

emitter operating in the negative-ion mode to generate deprotonated molecules

(anions) [110]. These even-electron anions can only react with analyte cations by

proton transfer. Thus it is necessary to trap these anions in a separate region in the

LTQ, where they first undergo a charge-selective CAD process to generate ETD-

inducing anion radicals, before they are allowed to react with analyte ions. This front-

end ETD approach has a major drawback in that a long acquisition time is required to

accommodate the additional CAD step and the switching delay between two pulsed

nESI sources. The newer commercial design employs an nCI source mounted on the

back of the orbitrap to generate radical reagent anions that can be brought into theLTQ

from its rear entrance. High-throughput ETD analyses on the chromatographic time

scale are readily achieved with this rear-end ETD design [111].

Ion fragmentation can also be achieved outside of the LTQ. For example, CAD can

be performed in the C-trap, by accelerating the isolated precursor ions from the LTQ

through the transfer octopole toward the C-trap [108]. The higher energy C-trap

dissociation can generate additional fragment ions that are unobtainable in the low-

energy CAD performed in the LTQ. During the C-trap CAD, however, a higher RF

amplitude is needed to trap efficiently the high-mass incoming precursor ions, leading

to an increase of the low-mass cutoff of the fragment ions. To overcome this difficulty,

the newer LTQ-Orbitrap XL instrument employs a dedicated collisional octopole

attached to the rear end of the C-trap for higher energy, collision-induced dissociation

(HCD). After dissociation in the collisional octopole, fragment ions are sent back to

the C-trap, and injected into the orbitrap for mass analysis. The C-trap CAD andHCD

tandemmass spectra often contain more structural information than low-energy CAD

spectra obtained in theLTQ, particularly in the lowm/z region. These low-m/z ions are

useful in a number of applications. For example, the presence of phosphotyrosine

immonium ions in higher energy CAD spectra provides information on PTMs [108].

As another example, quantitative proteomics studies employing iTRAQ labels cannot

be performed on ion trap instruments because the reporter ions are usually of too low

an m/z to be trapped efficiently, but iTRAQ can be performed on an LTQ-Orbitrap

with HCD capability [112].

The availability of these tandem MS tools, along with orbitrap’s superior mass

analysis performance and its compatibility to LC-MS/MS analysis, make the LTQ-

Orbitrap an extremely versatile and powerful mass spectrometer for a wide variety of

applications.

2.3.5 Ion-Mobility Instruments

To conclude this discussion on mass analysis and MS/MS in various instrument

configurations, we bring to the readers’ attention a new capability for MS. Ion

mobility has been known for some time, and when an ion-mobility device is

combined with a mass spectrometer, it gives a new dimension to peptide and

protein analysis. While it is still too early to determine the ion-mobility device’s

impact, we know that it offers the capability to do fast separations prior to mass


analysis and to investigate ion conformations, which are of key importance in the

biophysics of peptides and proteins.

Most mass spectrometers operate at 10�6 to 10�9 mbar background gas pressure

so that the perturbations on ion motion caused by collisions with neutral gas

molecules do not greatly affect their trajectories. Ion-mobility devices operate on

a different principle, at around 1mbar. Ions in an ion-mobility device are subjected

to a constant electric field at this high pressure so that they accelerate and quickly

achieve a terminal velocity, relying on the “drag” force from background gas

collisions to separate ions based on the balance between their acceleration (depen-

dent on their mass/charge ratio) and the drag (dependent on their cross-sectional

area and the mass of the background gas). Thus ion-mobility devices are not strictly

mass analyzers, the separation of the masses is also dependent on average cross-

sectional area [113,114]. Ion-mobility devices, however, have become quite useful

as quick gas-phase separation tools when combined with traditional mass analyzers

such as time-of-flight instruments [114–116].

For example, recently a mass-spectrometer manufacturer, Waters, released a new

instrument, called the Synapt, that employs an ion-mobility separator prior to a

quadrupole/time-of-flight instrument. In this case the ion-mobility separator uses a

stacked-ring geometry ion guide to keep the beam radially confined, but is still clearly

capable of separating ions by their rotationally averaged cross-sectional area. When

combined with a high-speed quadrupole/time-of-flight instrument, there are substan-

tial improvements in peak capacity, baseline chemical noise levels, and the ability to

perform conformation-dependent MS and tandem MS experiments, thus providing

interesting new capabilities. Other instrument manufacturers are also currently

working to develop similar, competing instruments.

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